1. Field of the Invention
The present invention generally relates to nucleic acid hybridization methods.
2. Description of the Related Art
Hybridization of polynucleotides to other polynucleotides by Watson-Crick base pairing is a fundamental process useful in a wide variety of research, medical, and industrial applications. Detecting the hybridization of a probe to a polynucleotide containing a target sequence is useful for gene expression analysis, DNA sequencing, and genomic analysis. Particular uses include identification of disease-related polynucleotides in diagnostic assays, screening for novel target polynucleotides in a sample, identification of specific target polynucleotides in mixtures of polynucleotides, identification of variant sequences, genotyping, amplification of specific target polynucleotides, and therapeutic blocking of inappropriately expressed genes, e.g. as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. (Cold Spring Harbor Laboratory, New York, 1989); Keller and Manak, DNA Probes, 2nd Ed. (Stockton Press, New York, 1993); Milligan et al., 1993, J. Med. Chem. 36: 1923-1937; Drmanac et al., Science, 260: 1649-1652; Bains, 1993, J. DNA Seq Map, 4:143-150.
Immobilized probes are useful for detecting polynucleotides containing a target nucleotide sequence, where each immobilized probe is functionally connected to a support and the hybridization of a polynucleotide to the immobilized probe can be detected. Most commonly, DNA probes are used to detect polynucleotides containing a target nucleotide sequence complementary to the probe sequence. The support for immobilized probes may be a flat surface, often called a “chip,” or the support may be the surface of a bead or other particle. Probes are usually immobilized in a known arrangement, or array, which provides a medium for matching known and unknown polynucleotides based on nucleic acid hybridization. Microarrays having a large number of immobilized probes of known identity are used to determine complementary binding, allowing massively parallel studies of gene expression and gene discovery. For example, an experiment with a single DNA chip can provide researchers information on thousands of genes simultaneously. For example, Hashimoto et al. disclose an array of immobilized single-stranded probes wherein at least one probe has a nucleotide sequence complementary to the target gene(s) to be detected, such that each probe is immobilized onto the surface of an electrode or the tip of an optical fiber and an electrochemically or optically active substance capable of binding to double-stranded nucleic acids is used to detect hybridization of target genes to complementary immobilized probes (U.S. Pat. Nos. 5,776,672 and 5,972,692).
Nonetheless, probe-based assays have serious limitations that are, at least partially, the result of difficulties associated with specificity, sensitivity and reliability.
At the simplest level, hybridization efficiency between a target nucleic acid and a capture probe is determined by the length of the complementarity between the target and the capture probe, the concentration of the target, and the temperature and the ionic strength of the reaction. It is also influenced by the sequence complexity of the complementary regions, and specifically, by the number of hydrogen bonds which will form between the target and the capture probe. Conventional methods for discriminating a single base pair mismatches in nucleic acid hybridization assays have focused on controlling parameters affecting the stringency of hybridization conditions, such as salt concentration and hybridization temperature. The stability of DNA hybrids in a solution increases as the concentration of cations in the solution increases, due to the ability of the cations to electrostatically shield the anionic phosphate groups in the DNA backbones from each other. Accordingly, hybridization conditions that have higher concentrations of cations, e.g. 5×SSC (0.75M NaCl, 0.075M Sodium Citrate), result in favor the kinetics of hybridization between complementary nucleic acids as well as between mismatched nucleic acids. Because the hybridization kinetics for duplexes is accelerated regardless of DNA sequence, performing the hybridization assays in high salt conditions by itself will not enhance discrimination between hybrids that are fully complementary and those that contain mismatches. On the other hand, allowing the hybridization assays to proceed in low salt conditions (e.g. 0.8×SSC (0.12M NaCl, 0.012 M Sodium Citrate) can enhance the discrimination between fully complementary nucleic acid hybrids and those that contain mismatches. However, the kinetics of hybridization is slowed in conditions of low salt, which in turn adversely affects the sensitivity of the hybridization assay (i.e., the lowest concentration of target DNA which will still yield a positive result). Sequence differences as subtle as a single base (point mutation) in very short oligomers (<10 base pairs “bp”) can be sufficient to enable the discrimination of the hybridization to complementary nucleic acid target sequences as compared with non-target sequences. Nonetheless, nucleic acid probes of greater than 10 bp in length are generally required to obtain the sequence diversity necessary to correctly identify a unique organism or clinical condition of interest. However, the ability to discriminate between closely related sequences is inversely proportional to the length of the hybridization probe because the difference in thermal stability decreases between wild type and mutant complexes as the probe length increases. Consequently, the power of probe based hybridization to correctly identify the target sequence of interest from closely related non-target sequences (e.g. targets harboring point mutations) poses several challenges.
In a review entitled “Principles and Practices of Nucleic Acid Hybridization” Kennel et al. teach the use of competitor RNA in a hybridization assay to estimate the specificity of the assay. David E Kennell, Principles and Practices of Nucleic Acid Hybridization, pp. 259-301, hereby expressly incorporated by reference in its entirety. The methods disclosed in Kennel et al. are based on the principle that two identical molecules will compete with each other for a common binding site. Kennel et al. apply this principle to assess similarities between two RNA populations competing for a common DNA. Typically, one population of RNA is labeled and the competitor population of RNA is unlabeled. The competition assay is used to estimate the degree of relation between the two RNA species. A process called “presaturation competition”, wherein the unlabeled competitor RNA is hybridized to the DNA before hybridization of the labeled RNA, has been reported to be useful in improving the results of this type of assay. However, the author warns that “great caution should be exercised” in interpreting the data from these assays.
U.S. Pat. No. 5,447,841 to Gray et al. describes methods of in situ hybridization of chromosomal DNA that involve decreasing the ability of labeled nucleic acid fragments to hybridize to repetitive sequences within the chromosomal DNA. Gray et al. disclose blocking the repetitive sequences within the chromosomal DNA by pre-reassociation of fragments with fragments of repetitive-sequence-rich DNA, by pre-reassociation of target DNA with fragments of repetitive-sequence-rich DNA, or pre-reassociation of both the fragments of the heterogeneous mixture and the target DNA with repetitive-sequence-rich DNA. According to Gray et al., this method provides blocking sufficient to permit detection of large labeled nucleic acid (greater than 1000 bp) hybridized to chromosomal DNA.
Methods, kits and compositions that improve the specificity, sensitivity and reliability of probe-based assays would be useful in the detection, analysis and quantitation of nucleic acid containing samples. There exists a need for an alternative to controlling the stringency of hybridization, e.g. with low salt or high temperature, to enable the discrimination of single base pair mismatches in nucleic acid hybridization assays, which maintaining sufficient detection sensitivity at low concentrations of amplicons.